Neonatal Gut Microbiome Composition and Gut-Derived Bloodstream Infections in a Cohort of Premature Singaporean Infants

Abstract

Abstract Background Due to their gestational age, low birth weight, and immature gut barriers, premature neonates are at high risk of bloodstream infections (BSI) caused by bacteria that reside in the gastrointestinal tract. Relatively little is known about the effect of alterations to the gut microbiota on the risk of BSI in premature neonates. Methods This prospective cohort study was conducted in the neonatal intensive care unit in the KK Women’s and Children’s Hospital in Singapore between June 1, 2019 and May 31, 2021. Clinical data and twice weekly fecal samples were collected during the first 75 days of life from infants born at or below 30 weeks gestational age. All infants received a Bifidobacterium breve probiotic starting at median (interquartile range [IQR]) day of life (DOL) 2 (1, 3). Fecal samples underwent shotgun metagenomic sequencing, and the resulting reads were aligned to Kraken 2 for taxonomic classification. We utilized permutational multivariate analysis of variance (PERMANOVA) and mixed effects linear regression to evaluate associations between clinical factors and gut microbiota composition. We described gut microbiota alterations among a subset of infants who developed a BSI. Results We collected 581 fecal samples from 75 neonates. Median (IQR) gestational age was 27 (25, 29) weeks and birthweight was 955 (763, 1210) grams. There were 18 BSIs that occurred among these infants and were caused by Streptococcus agalactiae (n=5), Klebsiella pneumoniae (n=3), Escherichia coli (n=2), Staphylococcus aureus (n=2), Staphylococcus epidermidis (n=2), Proteus mirabilis (n=1), Acinetobacter baumannii (n=1), Streptococcus anginosus (n=1), and Streptococcus pasteurianus (n=1). Metagenomic sequencing generated a median (IQR) sequencing depth of 5.9M (4.4M, 34.9M) paired-end reads per sample. The clinical factors that accounted for the most variance in gut microbiota composition were DOL (PERMANOVA; R2=0.022, p=0.001), feeding modality (R2=0.020, p=0.001), and antibiotic exposure (R2=0.018, p=0.001). Probiotic exposure was associated with an increase in the probiotic species, B. breve, and also a decrease in the relative abundances of several Clostridia species. Broad spectrum antibiotic exposures were associated with lower relative abundances of both potential pathogens, e.g., bacteria from the genera Enterobacter, Klebsiella, and Enterococcus, and beneficial bacteria, e.g., Bifidobacteria species, including the probiotic species. Notably, metronidazole and carbapenem exposures were associated with an increase in Staphylococcus species. Of 18 neonates who developed BSI, the causative species was detected in the gut microbiota of 17 infants. The relative abundance of the BSI species was ≥50% immediately prior to or at the time of BSI in 9 (50%) infants (Figure). Interestingly, four (22%) infants had a continued high abundance of the BSI species in the gut microbiota despite definitive antibiotic therapy. Conclusion Several clinical factors contribute to the gut microbiota composition of premature Singaporean neonates, especially postnatal age, feeding modality, and antibiotic exposures. Broad-spectrum antibiotic exposures affected the relative abundance of the probiotic agent and were associated with increases in staphylococcal species known to be potentially pathogenic in neonates. If detected in the gut microbiota, the bacteria that caused the BSI was often present in significant relative abundances both before and after the BSI. Figure. Relative abundances of bacterial species in the gut microbiota before and after bloodstream infections (BSI) in a subset of premature neonates. The relative abundances of the BSI species are plotted in color, whereas relative abundances of other detected species are depicted in gray. Each darkened vertical bar corresponds to a single fecal sample. Shading between each bar is provided to visualize changes in relative abundance between fecal samples. Black vertical lines depict the day of BSI. Specific antibiotic exposures are shown in the colored horizontal bars atop each subject’s time-series plot.